Methylation is the most abundant modification of DNA and RNA. It also represents one of the most important chemical signals in biology. The AlkB family proteins are newly discovered enzymes that use a novel oxidative demethylation process to remove a methyl group from DNA/RNA bases. The prototype, the E. coli AlkB, is a DNA/RNA base damage repair enzyme that catalyzes direct reversal of N1-methyladenine (1-meA), N3-methylcytosine (3-meC), and exocyclic DNA base lesions. Nine sequence homologues have been identified in the human genome. Some of these proteins play critical roles in DNA/RNA repair, obesity/diabetes, and various cancers. The oxidative demethylation mechanism is also used by other human proteins that are involved in key epigenetic processes such as histone demethylation and 5-meC hydroxylation in DNA. In this renewal application, we propose a combination of chemical, structural, and biological approaches to reveal the detailed demethylation mechanism and biological functions associated with the AlkB family proteins. We have developed and applied a chemical cross-linking strategy to stabilize labile and transient protein-DNA interactions of the AlkB family proteins. With the use of this strategy we will trap and characterize oxidative repair intermediates which should give clear mechanistic pictures for these as well as related oxygenases. We also propose to characterize protein-DNA interactions in the absence of DNA damage, thus providing further insight into the damage-searching mechanism used by these proteins. Both solution and theoretic methods will be employed to complement the structural study. The cross-linking strategy, proven successful in characterizing AlkB-DNA and ABH2- DNA complexes, will be applied to study other AlkB human homologues, in particular, ABH3, a prostate cancer marker, and FTO, a major factor involved in obesity and energy homeostasis. In addition, we have developed a photoactive unnatural amino acid that can be incorporated site-specifically onto proteins in live mammalian cells with high efficiency and fidelity. Photocross-linking with this new probe will allow us to pull down and identify specific partner proteins or substrates of ABH3 and FTO. Lastly, small molecule inhibitors for FTO will be developed, which can be used to further dissect the cellular function of FTO and serve as potential therapeutic leads to modulate human energy homeostasis. PUBLIC HEALTH RELEVANCE: The AlkB family proteins use a novel oxidative dememthylation mechanism to remove a methyl group from methylated DNA/RNA bases. Human AlkB homologues play key roles in DNA repair, obesity, and various cancers. The proposed research will lead to a better understanding of the demethylation mechanism and biological functions associated with these proteins.